The overall objective of this application is to understand the mechanism of RNA synthesis of mouse hepatitis virus (MHV-JHM), a murine coronavirus. Coronaviruses are a large family of RNA viruses which cause respiratory, gastrointestinal and neurological diseases in a variety of animals including humans. Coronavirus RNA synthesis is proposed to occur by a discontinuous mechanism which is not fully understood. Coronaviruses also undergo discontinuous copy-choice RNA recombination at a high frequency. To understand these unusual discontinuous mechanisms of coronavirus RNA synthesis, we will analyze the enzyme responsible for viral RNA synthesis, the RNA -dependent RNA polymerase. The coronavirus RNA polymerase is synthesized as a large precursor polyprotein which must be processed into functional subunits. We hypothesize that a cascade of proteolytic processing events regulates the activity and specificity of the coronavirus RNA polymerase. We propose to identify the processed subunits of the polymerase by generating antibodies to specific domains of the RNA polymerase, and using those antibodies to identify viral proteins from infected cells. To determine how the RNA polymerase polyprotein is processed into subunits, we are generating polymerase polyprotein products in a cell-free system and monitoring the translation products for proteolytic activity. The identify the function of the components of the RNA polymerase polyprotein, we are developing in vitro assays for activity and identifying specific mutations which result in the loss of RNA synthesis in temperature sensitive mutants of coronavirus. We will also determine if host factors are required for RNA synthesis or RNA stability by testing coronavirus replication when host factors are inhibited or absent. Overall, these studies are designed to identify functional domains of the coronavirus RNA- dependant RNA polymerase. If we can understand how the coronavirus RNA -dependent RNA polymerase is generated, and how it transcribes and replicates the viral genetic information, we can design specific drugs to block viral replication and pathogenicity.